ALBERT

Description: Age-group analyses were conducted of patients in the prophylactic platelet dose trial (PLADO), which evaluated the relation between platelet dose per transfusion and bleeding. Hospitalized patients with treatment-induced hypoproliferative thrombocytopenia were randomly assigned to 1 of 3 platelet doses: 1.1 × 1011, 2.2 × 1011, or 4.4 × 1011 platelets/m2 per transfusion, given for morning counts of ≤ 10 000 platelets/μL. Daily hemostatic assessments were performed. The primary end point (percentage of patients who developed grade 2 or higher World Health Organization bleeding) was evaluated in 198 children (0-18 years) and 1044 adults. Although platelet dose did not predict bleeding for any age group, children overall had a significantly higher risk of grade 2 or higher bleeding than adults (86%, 88%, 77% vs 67% of patients aged 0-5 years, 6-12 years, 13-18 years, vs adults, respectively) and more days with grade 2 or higher bleeding (median, 3 days in each pediatric group vs 1 day in adults; P 〈 .001). The effect of age on bleeding differed by disease treatment category and was most pronounced among autologous transplant recipients. Pediatric subjects were at higher risk of bleeding over a wide range of platelet counts, indicating that their excess bleeding risk may be because of factors other than platelet counts. This trial was registered at www.clinicaltrials.gov as #NCT00128713.

Description: Background : S-303 treatment (200 μM S-303 and 2 mM glutathione) has been shown to inactivate viruses, bacteria, protozoa and leukocytes in RBC concentrates. S-303-treated RBCs (SRBC) have comparable in vitro parameters to untreated conventional RBCs (CRBC) over 42 days (d) of storage. Three Phase I trials in healthy human subjects showed recovery and life span of 35 d old autologous S-303 RBC were comparable to that for CRBC without detectable antibody (Ab) formation. During the course of a Phase III trial for patients (pts) in chronic transfusion (txn) programs receiving repeated SRBC txn, 2 pts developed asymptomatic anti-SRBC Ab, leading to trial termination. Methods : A randomized, controlled, double-blinded, crossover design, non-inferiority trial of SRBC in pts with thalassemia (thal) or sickle cell anemia (SSA) was conducted. Fifty pts were to receive a course (≥5 months or ≥6 txn) of SRBC or CRBC with crossover to the other arm. The frequency and number of RBC units per txn and target hemoglobin (Hb) were at physician discretion. The primary endpoint was Hb txed per kg body weight per d. Secondary endpoints were: time between txn, pre-txn Hb, number of RBC units txed, Ab formation, and adverse events (AEs). Routine DAT and IAT with RBC panels, and cross match with IAT to SRBC (and to the pre-S-303 treatment segment if positive to SRBC), were performed at trial sites prior to each txn. Following detection of positive cross match to SRBC in 2 pts, all pre-txn samples from all pts were tested by polyethylene glycol (PEG) SRBC cross match with IAT at a reference lab. Post study termination, pts had monthly follow-up (for up to 6 months) for AEs and repeated SRBC PEG cross match IAT. Results : The trial was terminated after 26 pts received ≥1 txn when 2 asymptomatic pts in the SRBC arm developed positive (+) pre-txn IAT cross matches with SRBC but not the same RBC unit pre-S-303 treatment (SRBC Ab). No pt completed the trial; 69% started the 1st period only and 31% crossed over to the 2nd period. 17 pts had ≥1 SRBC txn. Neither pt with (+) cross matches had clinical evidence of significantly reduced RBC survival. Hapten inhibition assays showed inhibition by S-303 derivatives but not glutathione used in the treatment process; a monocyte monolayer RBC phagocytic assay was negative with pt sera, suggesting these Ab were not likely clinically significant. Six-month follow-up of all txed pts did not reveal additional SRBC Ab or AEs. No Ab to SRBC were detected in a companion Phase III trial of SRBC txn for pts undergoing cardiac surgery supported with SRBC txn for up to 7 d. Conclusions : Two of 17 pts with exposure to SRBC in chronic txn programs developed Ab to SRBC after txn with SRBC. These Ab did not appear to result in decreased RBC survival (e.g., hemolysis), AEs, autoantibody formation, or Ab formation to RBC antigens. None of 74 pts txed with SRBC for up to 7 d to treat anemia of cardiac surgery developed SRBC Ab. A modified S-303 treatment process to reduce immunogenicity has been developed. Patients With Antibodies to S-303 RBC Patient 1 Patient 2 Age (yr) 3 13 Sex F M Diagnosis Thal SSA Time on study (d) 139 58 AEs Splenomegaly Pt. blood type AB+ A+ DAT/IAT (allo-Ab panel RBC) −/− −/− No. SRBC txn/units before (+) crossmatch 5/10 1/1 IAT reaction (LISS/PEG) against SRBC 2+/3+ 3+/2+ Titer of (+) sera against SRBC 2 8

Description: Key Points Overall, no benefit of granulocyte transfusion therapy was observed, but the power of the study was reduced due to low accrual. Post hoc secondary analysis suggested that patients receiving higher doses tended to have better outcomes than those receiving lower ones.

Description: Cold agglutinin disease (CAD) is an autoimmune hemolytic anemia in which autoantibodies bind to red blood cells (RBC) at temperatures below 37°C, resulting in activation of the classical complement pathway (CCP). CCP activation leads to hemolysis either intravascularly, by formation of the membrane attack complex, or extravascularly, when C3/C4 fragment deposition onto the RBC surface results in sequestration by the reticuloendothelial system. Here we describe the in vitro and in vivo activity of TNT003 and TNT009, inhibitors of a serine protease specific to the CCP, in pre-clinical models of CAD. TNT003 is a mouse monoclonal IgG2a antibody with sub-nanomolar affinity. TNT009 is the humanized form (IgG4) of TNT003 and retains affinity and specificity to its target. In vitro assays using IgM-sensitized sheep RBC and human or non-human primate (NHP) serum showed that TNT003 and TNT009 potently inhibited antibody-mediated hemolysis in a concentration dependent manner. Additionally, TNT003 and TNT009 inhibited CCP-mediated production of the anaphylatoxins C4a, C3a, and C5a. Flow cytometry analysis showed that both antibodies also prevented C3 fragment deposition on the RBC surface. Activity was proportional to the amount of serum used, and at 80% human or NHP serum, TNT003 completely inhibited hemolysis with an IC50 of ∼13 µg/mL. Using an ELISA-based assay, TNT003 inhibited C5b-9 deposition driven by the CCP but not by the alternative (CAP) or lectin (CLP) pathways. These data suggest that TNT003 and TNT009 are specific and potent inhibitors of the CCP. To demonstrate the utility of a CCP inhibitor in disease, we tested the ability of TNT003 and TNT009 to inhibit the CCP in ex vivo hemolysis assays using CAD patient autoantibodies. Type O- RBC were incubated in the presence of CAD plasma to sensitize the cells with autoantibody. RBC were then washed and 25% normal human serum (NHS) added as a source of complement. Thirteen of the seventeen CAD samples tested (76%) mediated C3 fragment deposition on the RBC surface as determined by flow cytometry. TNT003 significantly inhibited C3 fragment deposition by all patient samples that deposited complement (88 ± 2.6% inhibition, n = 13) with an average IC50 of 4.7 ± 0.4 µg/mL. One patient sample induced complement-dependent hemolysis of ∼50% of the RBC upon addition of NHS. In a concentration dependent manner, TNT003 and TNT009, but not control IgG, completely inhibited CAD autoantibody-mediated hemolysis (Fig. 1), as well as C4a, C3a and C5a generation. We further characterized each patient sample to determine cold agglutinin titer. We found that cold agglutinin titer correlated with the percent RBC staining positive for cell surface C3 fragments (R2 = 0.3566; p 〈 .01; n = 17 samples; Fig. 2).Figure 1TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 1. TNT003 and TNT009 inhibit CAD autoantibody-mediated hemolysisFigure 2Cold agglutinin titers correlate with C3 fragment deposition on RBCFigure 2. Cold agglutinin titers correlate with C3 fragment deposition on RBC Extravascular hemolysis of C3 fragment-coated RBC by liver macrophages is believed to be the primary mechanism of RBC destruction in CAD. We therefore tested the hypothesis that CAD patient plasma-induced C3 fragment deposition on RBC would promote phagocytosis by the monocytic cell line THP-1. We found that RBC sensitized in CAD plasma and exposed to NHS were engulfed in an FcgR-independent mechanism by THP-1 cells. RBC phagocytosis was significantly inhibited if NHS exposure occurred in the presence of TNT003 (100 µg/mL), but not a control IgG. The selective CCP inhibitory activity of TNT003 was evaluated in vivo in cynomolgus monkeys. TNT003 administered as a single IV injection at 30 mg/kg resulted in a Cmax of ∼330 µg/mL and detectable serum TNT003 thru ≥72 hours. Using an ELISA-based assay, we observed specific inhibition (≥95%) of the CCP for ≥72 hours. In contrast, CAP activity was modestly and transiently inhibited for 4 - 8 hours. At Cmax, endogenous C4a levels were reduced by 〉90% and returned to baseline levels by ≥96 hours. Serum samples containing TNT003 showed complete (100%) inhibition of hemolysis and C3 fragment deposition in vitro. CCP activity was completely restored to baseline after TNT003 concentrations fell below a predictable, threshold level. Collectively, these data indicate that TNT003 and TNT009 are potent and specific inhibitors of CCP activity and C3 fragment deposition in vitro and in vivo. These findings support the preclinical development of TNT009 for the treatment of CCP-mediated diseases including CAD. Disclosures: Panicker: True North Therapeutics, Inc.: Employment, Equity Ownership. Shi:True North Therapeutics, Inc.: Employment, Equity Ownership. Rose:True North Therapeutics, Inc.: Employment, Equity Ownership. Hussain:True North Therapeutics, Inc.: Employment, Equity Ownership. Tom:True North Therapeutics, Inc.: Employment, Equity Ownership. Strober:True North Therapeutics, Inc.: Employment. Sloan:True North Therapeutics, Inc.: Consultancy. Parry:True North Therapeutics, Inc.: Employment, Equity Ownership. Stagliano:True North Therapeutics, Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees.

Description: More than 1 million apheresis platelet collections are performed annually in the United States. After 2 healthy plateletpheresis donors were incidentally found to have low CD4+ T-lymphocyte counts, we investigated whether plateletpheresis causes lymphopenia. We conducted a cross-sectional single-center study of platelet donors undergoing plateletpheresis with the Trima Accel, which removes leukocytes continuously with its leukoreduction system chamber. We recruited 3 groups of platelet donors based on the total number of plateletpheresis sessions in the prior 365 days: 1 or 2, 3 to 19, or 20 to 24. CD4+ T-lymphocyte counts were

Description: Abstract SCI-47 Transfusion medicine has long recognized that there is significant variation in the human immune response to red blood cell (RBC) transfusions.1 All allogeneic RBC transfusions expose the recipient to multiple foreign antigens, but very few RBC transfusions induce a detectable humoral immune response. Using statistical analyses of antibody responses among transfused patient populations, we found that the response closely approximates a geometric distribution that would be predicted by a random memory-less (stochastic) process. These statistical analyses demonstrate that most patients fail to mount immune responses to RBC transfusions but a distinct group of patients, termed potential responders, is capable of mounting an immune response to blood transfusions. Potential responders comprise approximately 13% of the transfused population and this proportion does not depend on pathologic diagnoses, age, gender or racial group. The only group that has a higher rate of potential responders are pregnant women, but those patients have a second route of antibody exposure. Although the ability to immunologically respond to blood transfusions appears to be limited to the relatively small population of potential responders, even potential responders do not mount an immune response to most RBC transfusions. Additionally, the specific antibodies that patients make are not completely independent of each other. Indeed, patients who have made some specific antibodies are more likely to subsequently make one set of anti-erythrocyte antibodies and less likely to make another set of anti-erythrocyte antibodies. This suggests that the potential immune repertoire differs between patients and the difference in potential repertoire is not solely explained by the antigens expressed on the patients' erythrocytes. Moreover, the potential repertoire is influenced by the age of the patient. Younger patients have a more diverse immune response producing antibodies against a broad spectrum of RBC antigens. As patients get older, the immune response becomes more limited and usually only responds against the most immunogenic RBC antigens. In summary, these results suggest that potential responders to intravenous administration of RBCs comprise a genetically distinct group and that additional factors determine which transfusion will induce an immune response in potential responders. The factors that cause a person to be a potential responder and the factors that cause a potential responder actually respond to a specific transfusion are subjects of ongoing research. In the meantime, transfusion services may consider minimizing immunologic stimuli to patients who are clearly responders. 1. Higgins JM, Sloan SR. Stochastic modeling of human RBC alloimmunization: evidence for a distinct population of immunologic responders. Blood. 2008;112:2546-2553. Disclosures No relevant conflicts of interest to declare.